1. Field of the Invention
The invention relates to DNA sequencing.
2. Brief Description of the Prior Art
The sequence analysis of large DNA molecules is possible since the development of the Maxam-Gilbert and Sanger-Coulson methods. These methods are based on three main principles:
(1) The use of a labelled nucleoside triphosphate in vitro for the radioactive labelling of the polynucleotide at its 5' or 3' ends or therebetween;
(2) The use of a cutting technique on the polynucleotide to be sequenced to form four or more groups of fragments with a fixed common end;
(3) The use of a polyacrylamide gel electrophoresis in order to sort, according to length, molecules within the groups, and copy them.
The nucleotide sequence can then, by mutual comparison of the different fractionation patterns of the four groups, be read in the form of an autoradiograph of the polyacrylamide gel. Each individual analysis can produce the sequence of a DNA segment of 200 to 500 nucleotides. These individual segments are then classified by examining overlapping stretches or areas.
A sequence analysis depends chiefly on ease of production of the fragments, which are analysed in suitable form before the base-specific reactions. In the Sanger-Coulson method this problem is solved by subcloning random DNA fragments in vectors derived from bacteriophage M13 at an insertion site in the vicinity of a sequence specific to the vector, from which sequencing reactions proceed (primer initiated repair synthesis in the presence of dideoxytriphosphates). The single-strand DNA matrix can be isolated by simple manipulations.
By contrast, the Maxam-Gilbert method require one or more gel separation stages and fragment-specific enzymatic reactions for the introduction and separation of the radioactive labelling. Consequently, the strategy often depends on an already existing arrangement of restriction cleavage sites in order to reduce the number of individual analyses.
Plasmid subcloning systems similar to the M13 system have been developed in various laboratories. Their object is the use of chemical degradation sequencing of individual recombinant subclones of a DNA molecule that is to be sequenced. The basic feature of these sequencing vectors is that two restriction cleavage sites are in the vicinity of the subcloning site (insertion site). The site in closer proximity to the insertion is used for the labelling reaction, whilst the other site permits separation of the labelling. Thus two labelled fragments are formed. The smaller fragment is not removed and produces a non-readable sequence in the lower region of the sequencing gel. This loss of sequence information amounts to fewer than 30 nucleotides in all of the processes hitherto known. All of these processes still include at least two restrictions and one labelling reaction.
The object of the present invention is to introduce DNA to be sequenced, or fragments of DNA to be sequenced, into a double-stranded vector, which can be individually labelled in a simple manner. It is intended, therefore, that only one strand of the vector be labelled.